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Method for mass production of pseudorabies virus vaccine

A pseudorabies virus, large-scale technology, applied in biochemical equipment and methods, tissue cell/virus culture devices, antiviral agents, etc., can solve the monitoring and control of environmental conditions, high labor intensity, low cell density, etc. problems, to achieve uniform and stable product quality, reduce the probability of contamination, and increase the effect of virus content

Inactive Publication Date: 2010-08-18
PU LIKE BIO ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, in the production of biological products in my country, anchorage-dependent cells such as African green monkey kidney cells (Vero) and pig kidney cells (PK15) are mostly cultured in traditional spinner bottles, but the area where the cells can proliferate is limited to culture. Due to the limited area of ​​the bottle, it is difficult to monitor and control the environmental conditions of the culture, so the cell density is low, and the labor intensity is high, the operation process is easy to be polluted, and the vaccine production and quality are greatly limited.
At present, the problems in the production of pseudorabies vaccine are as follows: 1. The labor intensity is high and the floor space is large; 2. The cell growth area provided per unit volume is limited and the cell density is low; big difference

Method used

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  • Method for mass production of pseudorabies virus vaccine
  • Method for mass production of pseudorabies virus vaccine
  • Method for mass production of pseudorabies virus vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Large-scale production of pseudorabies virus vaccine

[0027] The bioreactor used in this example is the TideCell-020 type produced by CESCO Company in the United States, the volume of the carrier tank is 20L, and the volume of the culture medium bag is 500L. The carrier used is BioNoc II polyester fiber produced by the company. The fiber is a network structure, which is hydrophilic and biologically harmless. 1g of carrier provides about 2400cm 2 The adherent area of ​​​​can provide about 1.0 x 10 9 Cell growth space requires 11 g of BioNoc II polyester fiber per liter of carrier tank volume.

[0028] (1) Preparation of porcine kidney cell (PK15) seed solution for seedling production to make the total number of cells reach 5×10 9 .

[0029] (2) Add the PK15 cell seed solution described in step (1) to the carrier tank containing the carrier, and add 500L DMEM (Dulbecco's modification of Eagle's medium Dulbecco) cell growth solution containing 5% bovine serum...

Embodiment 2

[0037] Example 2 Test of pseudorabies virus vaccine

[0038] (1) Character test: light yellow spongy loose agglomerates, dissolved quickly after adding PBS (0.01mol / L pH7.2), and showed a uniform suspension.

[0039] (2) Pure inspection: In accordance with pages 15, 19 and 20 of the third appendix of the 2005 edition of the Veterinary Pharmacopoeia of the People's Republic of China, there is no bacterial, mold, mycoplasma or foreign virus contamination.

[0040] (3) Virus content: Dilute with PBS (0.01mol / LpH7.2) according to the bottle label, and measure according to the method for measuring virus content in step (6) of Example 1, the virus content of each head is ≥ 5000TCID 50 .

[0041] (4) Safety inspection: diluted with PBS (0.01mol / L pH7.2) according to the bottle label to contain 14 copies per 5ml, intramuscular injection of 2 sheep without pseudorabies virus neutralizing antibody from 6 to 18 months , 5ml per head, observed for 14 days, no clinical reaction. The res...

Embodiment 3

[0048] Example 3 Comparison of Suspension Culture Technology and Traditional Spinner Bottle Culture Technology

[0049] In the present invention, statistical analysis was done on TideCell and 3000ml spinner flask culture system, and the results are shown in Table 3.

[0050] Table 3 Correlative comparison of the proliferation of pseudorabies virus in different culture systems

[0051]

[0052] The volume of the carrier tank of the TideCell-020 suspension culture system used in the present invention is 20L, the carrier is BioNoc II polyester fiber, 220g of carrier is added, and 1g of carrier can provide 2400cm 2 To achieve the same adhesion area as TideCell-020, 423 3000ml spinner flasks are required, but the amount of antigen obtained is more than 3 times that of the spinner flask system. The entire TideCell adopts a fully automatic closed culture, which requires significantly less manpower than the spinner system, and the probability of antigen liquid contamination is als...

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Abstract

The invention discloses a method for mass production of pseudorabies virus vaccine, comprising the following steps: (a) adding netty polyester fiber which serves as a carrier into a bioreactor provided with a tide type micro-carrier suspension culture system and inoculating cells for producing vaccine; (b) inoculating pseudorabies virus vaccine when the culture cell grows to a certain intensity, so that the cells are infected by the pseudorabies virus vaccine; (c) reproducing the virus in great numbers under appropriate conditions; (d) harvesting the virus when cytopathic rate reaches above 70%; (e) carrying out freeze thawing on the harvested virus for once or twice to lead the cells to completely come off and disperse and then adding freeze-drying protective agent, evenly mixing the mixture, packaging the mixture in fixed volume and freeze-drying.The method of the invention has the advantages of good stability, explicit process control indicators, good controllability, easy operation, large process scale and the like.

Description

technical field [0001] The invention belongs to the technical field of biological products, and relates to a large-scale production method of pseudorabies virus vaccine. Background technique [0002] Pseudorabies virus (PRV) belongs to the alphaherpesvirus subfamily of the family Herpesviridae, and has the characteristics of rapid growth on cell growth, strong neurotropism and latent infection. The virus can infect pigs, cattle, sheep, dogs, cats, rabbits, mice, minks, foxes and other livestock and wild animals under natural conditions. Pigs are the reservoirs and transmitters of the virus, mainly manifested as abortion, stillbirth or mummified fetuses in pregnant sows, neurological symptoms, paralysis, failure and even death in newborn piglets, with a mortality rate of almost 100%, and the incidence of weaned piglets is 20-40%. %, and the mortality rate is 10-20%. The disease has become the main cause of reproductive dysfunction syndrome in breeding pigs and is increasing ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/245A61P31/22C12M3/02
Inventor 张许科孙进忠乔荣岑陶家权骆爱芳张海洋习向锋李三
Owner PU LIKE BIO ENG
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